Modified peptides and proteins are valuable biophysical tools for studying biological processes, both in vitro and in vivo. They are also useful in assays to identify new drugs and therapeutic agents. In particular, quantitative live cell imaging using fluorescent proteins and peptides is revolutionizing the study of cell biology. An exciting recent development within this field has been the construction of peptide and protein biosensors exhibiting altered fluorescence properties in response to changes in their environment, oligomeric state, conformation upon ligand binding, structure, or direct ligand binding. Appropriately labeled fluorescent biomolecules allow spatial and temporal detection of biochemical reactions inside living cells. See for example Giuliano, K. A., et al., Annu. Rev. Biophys. Biomol. Struct. 1995,24:405-434; Day, R. N. Mol. Endocrinol. 1998, 12:1410-9; Adams, S. R., et al., Nature 1991, 349:694; Miyawaski, A., et al., Nature 1997, 388:882-7; Hahn, K., et al., Nature 1992, 359:736; Hahn, K. M., et al., J. Biol. Chem. 1990,265: 20335; and Richieri, G. V., et al., Mol. Cell. Biochem. 1999, 192:87-94.
Procedures for site-specific modification of polypeptides have been described, including: chemically selective labeling in solution Brinkley, M. Bioconjugate Chemistry 1992, 3:2-13) and on resin bound peptides (Hackeng, T., et al., J. Biol. Chem. Submitted); introduction of ketone amino acids through synthetic procedures (Rose, K. J. Am. Chem. Soc. 1994, 116:30-33; King, T. P., et al., Biochemistry, 1986, 25:57745779; Rose, K., et al., Bioconjugate Chem. 1996, 7:552-556; Marcaurelle, L. A., Bertozzi, C. R. Tett. Lett. 1998, 39:7279-7282; and Wahl, F., Mutter, M. Tett. Lett. 1996, 37:6861-6864); and molecular biology techniques (Cornish, V. W., et al., J. Am. Chem. Soc. 1996, 118:8150).
While each of these methods has utility for producing a particular class of biosensor or labeled polypeptide, all have limitations that restrict their general use. Labeling of natural amino acid sidechains in solution is often impractical because of the existence of many other competing nucleophiles. Additionally, the use of unnatural amino acids, such as those bearing ketones for selective labeling, requires the synthesis of dye constructs or amino acids that are difficult to make and not available commercially.
Currently, the major obstacles to the development of fluorescent biosensors remain: (1) The difficulty in site-specific placement of the dye in the polypeptide and (2) determining exactly which site is optimal for dye placement (Giuliano, K. A., et al., Annu. Rev. Biophys. Biomol. Struct. 1995, 24:405-434). Solvent-sensitive dyes and other biophysical probes must be placed precisely for optimal response to changes in protein structure without interference with biological activity. Also, the need for site-specific incorporation of two dyes without impairment of biological activity has proven a serious limitation for utilization of fluorescence resonance energy transfer (FRET) within a single protein. Total chemical synthesis of proteins provides a potential solution to these problems (Wilken, J., Kent, S. B. H. Curr. Op. Biotechnology. 1998, 9:412; Kent, S. B. H. Ann. Rev. Biochem. 1988, 57, 957-989; Dawson, P. E., et al., Science 1994, 266:776-779; Muir, T. W., et al., Proc. Natl. Acad. Sci. 1998, 95:6705-6710; and Cotton, G. J., et al., J. Ann. Chem. Soc. 1999, 121:1100-1101). However, many biophysical probes suitable for fluorescent biosensors or other purposes are not stable to the various conditions used for peptide synthesis, and site-specific incorporation after synthesis is difficult to achieve.
Thus, there is currently a need for peptide synthons having protected functional groups that can be selectively modified to incorporate one or more functional molecules (e.g. a biophysical probe such as a fluorescent label) following peptide synthesis.